Diagnostic test elements are important components of clinically relevant analytical methods. In this connection, the focus is on the measurement of analytes, for example metabolites or substrates, which for example can be determined directly or indirectly with the aid of an enzyme that is specific for the analyte. In this case, the analytes are converted with the aid of an enzyme-coenzyme complex and subsequently quantified. This entails the analyte to be determined being brought into contact with a suitable enzyme, a coenzyme and optionally a mediator, whereby the coenzyme is physicochemically changed, for example oxidized or reduced, by the enzymatic reaction. If a mediator is additionally used, it usually transfers electrons from the reduced coenzyme released during the conversion of the analyte onto an optical indicator or the conductive components of an electrode so that the process can be detected, by way of non-limiting example, photometrically or electrochemically. A calibration provides a direct relationship between the measured value and the concentration of the analyte to be determined.
An important criterion when providing diagnostic test elements is their long-term stability. Certain test elements known from the prior art which are used in the determination of blood glucose are generally very sensitive to moisture and heat, such that upon exposure to the same the function of the coenzyme and mediator, for example, is usually impaired. Another problem of commercially available test elements is their sensitivity to ambient light where light absorption by the enzyme system can result in damage to the enzyme, coenzyme and/or mediator. In certain instances where, for example, tests are carried out by the end user himself, erroneous results can therefore occur due to an incorrect, unnoticed faulty storage of the measurement system which can be hardly detected by the user and may result in incorrect treatment of the respective disease.
A known measure that can be used to increase the stability of diagnostic test elements is the use of stable enzymes, including for example the use of enzymes from thermophilic organisms. Furthermore, it is possible to stabilize enzymes by chemical modification such as cross-linking, or by mutagenesis. In addition, enzyme stabilizers such as trehalose, polyvinyl pyrrolidone and serum albumin for example, can also be added or the enzymes can be enclosed in polymer networks by photopolymerization for example.
Attempts have also been made to improve the stability of diagnostic test elements by using stable mediators. Thus, the specificity of tests is increased and interferences during the reaction are eliminated by using mediators with a redox potential that is as low as possible. However, the redox potential of the enzyme/coenzyme complexes forms a lower limit for the redox potential of mediators. If the redox potential is lower than this limit, the reaction with the mediators is slowed down or even stopped.
Alternatively, it is also possible to use diagnostic test elements without mediators in which, for example, coenzymes such as the coenzyme NADH are detected directly. A disadvantage of such measurement systems is, however, that native coenzymes such as NAD and NADP are unstable.
NAD and NADP are base-labile molecules whose degradation pathways are described in the literature (N. J. Oppenheimer, in “The Pyridine Nucleotide Coenzyme”, Academic Press New York, London 1982, Editor J. Everese, B. Anderson, K. You, chapter 3, pages 56-65). ADP-ribose is mainly formed when NAD or NADP are degraded by cleavage of the glycosyl linkages between the ribose and the pyridine unit. In contrast, the reduced forms NADH and NADPH are acid-labile: for example, epimerization is a known degradation pathway. In both cases, the instability of NAD/NADP and NADH/NADPH is due to the lability of the glycosyl linkage between the ribose unit and the pyridine unit. However, under conditions that are not drastic such as an aqueous solution for example, the coenzymes NAD and NADP are already hydrolyzed solely due to the ambient moisture. This instability may lead to inaccuracies in the measurement of analytes.
A number of NAD/NADP derivatives is described, for example, by B. M. Anderson in “The Pyridine Nucleotide Coenzymes”, Academic Press New York, London 1982, editor J. Everese, B. Anderson, K. You, chapter 4. However, most of these derivatives are not well accepted by enzymes. The only derivative that has therefore been used up to now for diagnostic tests is 3-acetylpyridine adenine dinucleotide (acetyl-NAD) which was described for the first time in 1965 (N. O. Kaplan, J. Biol. Chem. (1956), 221, 823). This coenzyme also shows a poor acceptance by enzymes and a change in the redox potential.
International Patent Publication No. WO 01/94370 describes the use of further NAD derivatives with a modified pyridine group. However, modifications of the nicotinamide group generally have a direct influence on the catalytic reaction. In most cases this influence is negative.
In another stabilization concept, the ribose unit was altered in order to thus influence the stability of the glycosyl linkage. This procedure does not directly interfere with the catalytic reaction of the nicotinamide group. However, it may have an indirect influence as soon as the enzyme exhibits a strong and specific binding to the ribose unit. Kaufmann et al. disclose in this connection a number of thioribose-NAD derivatives in International Patent Publication No. WO 98/33936 and U.S. Pat. No. 5,801,006 and in International Patent Publication No. WO 01/49247. However, a relationship between the modification of the nicotinamide ribose unit and the activity of the derivatives in enzymatic reactions has not been shown to date.
carbaNAD, a derivative without a glycosyl linkage was described for the first time in 1988 (J. T. Slama, Biochemistry (1988), 27, 183, and Biochemistry (1989), 28, 7688). The ribose therein is substituted by a carbacyclic sugar unit. Although carbaNAD was described as a substrate for dehydrogenases, its activity has previously not been demonstrated clinically in biochemical detection methods.
A similar approach was described later by G. M. Blackburn (Chem. Comm. (1996), 2765) in order to prepare carbaNAD with a methylene bisphosphonate compound instead of the natural pyrophosphate. The methylene bisphosphonate shows an increased stability towards phosphatases and was used as an inhibitor for ADP-ribosyl cyclase. An increase in hydrolysis stability was not the aim (J. T. Slama, G. M. Blackburn).
International Patent Publication No. WO 2007/012494 and U.S. Pat. No. 7,553,615 finally disclose stabilized NAD/NADH and NADP/NADPH derivatives, enzyme complexes of these derivatives and their use in biochemical detection methods and reagent kits.
One non-limiting object of the present application is to provide a method for stabilizing enzymes, especially for the long-term stabilization of enzymes which at least partially eliminates the above-mentioned disadvantages.